Bottom Line:
Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW.The body of treated insects showed inhibition of sclerotization, leading eventually to death.Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing.

ABSTRACTThe African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

pone.0115336.g003: Effect of inhibition of laccase2 expression after injection with dsRNA in second-instar larvae of Cylas puncticollis.(A) Mortality after injection with dsRNA targeting laccase2 (dslac2) (day 14–20) expressed in percentage. Mortality in larvae injected with dsRNA targeting gfp (dsgfp) (control) was only 8% (B) Larvae injected with dsgfp as a control and (C) treated larvae after 3 days; (D) Pupa development 6 days after injection with dsgfp as a control and (E) dslac2; (F) Adult development injected 10 days after injection with dsgfp as a control (G) dslac2 (H) Surviving individual 13 days after injection with dslac2. Larvae were injected with the dsRNA solution into the hemocoel at a concentration of 0.2 µg/mg body weight. The insects were kept in sweetpotato roots after injection for the duration of the experiment.

Mentions:
Inhibition of laccase2 expression could be observed phenotypically in 21 of 25 (84%) individuals as early as 3 days following injection. Treated larvae exhibited lack of sclerotization in the head capsule resulting in an untanned cuticle (Fig. 3C.1) compared to the control larvae injected with gfp dsRNA (Fig. 3B.1). Injection trauma in the control resulted in 8% of mortality. Suppression of laccase2 expression can be detected after 24 h when tested by qPCR (Fig. 4). These results demonstrate that laccase2 mRNA levels were reduced 91.7% compared to the control injected with gfp dsRNA (p-value 0.0193) after 24 h. This reduction was also observed at the other two time points, where the expression levels on day 3 and day 5 showed a reduction of 92.9% (p-value 0.0107) and 93% (p-value 0.001), respectively. Interestingly, expression of laccase2 was found to be variable between different larval stages and even within a certain stage. Possibly, laccase2 expression only exhibits a peak at a certain time after the molt, given its role in the cuticle tanning. However, further studies should be conducted in order to confirm this. Nevertheless, despite this natural variability, the silencing we observed was strong when compared to the control for each time point. and consistent in all experiments and repetitions.

pone.0115336.g003: Effect of inhibition of laccase2 expression after injection with dsRNA in second-instar larvae of Cylas puncticollis.(A) Mortality after injection with dsRNA targeting laccase2 (dslac2) (day 14–20) expressed in percentage. Mortality in larvae injected with dsRNA targeting gfp (dsgfp) (control) was only 8% (B) Larvae injected with dsgfp as a control and (C) treated larvae after 3 days; (D) Pupa development 6 days after injection with dsgfp as a control and (E) dslac2; (F) Adult development injected 10 days after injection with dsgfp as a control (G) dslac2 (H) Surviving individual 13 days after injection with dslac2. Larvae were injected with the dsRNA solution into the hemocoel at a concentration of 0.2 µg/mg body weight. The insects were kept in sweetpotato roots after injection for the duration of the experiment.

Mentions:
Inhibition of laccase2 expression could be observed phenotypically in 21 of 25 (84%) individuals as early as 3 days following injection. Treated larvae exhibited lack of sclerotization in the head capsule resulting in an untanned cuticle (Fig. 3C.1) compared to the control larvae injected with gfp dsRNA (Fig. 3B.1). Injection trauma in the control resulted in 8% of mortality. Suppression of laccase2 expression can be detected after 24 h when tested by qPCR (Fig. 4). These results demonstrate that laccase2 mRNA levels were reduced 91.7% compared to the control injected with gfp dsRNA (p-value 0.0193) after 24 h. This reduction was also observed at the other two time points, where the expression levels on day 3 and day 5 showed a reduction of 92.9% (p-value 0.0107) and 93% (p-value 0.001), respectively. Interestingly, expression of laccase2 was found to be variable between different larval stages and even within a certain stage. Possibly, laccase2 expression only exhibits a peak at a certain time after the molt, given its role in the cuticle tanning. However, further studies should be conducted in order to confirm this. Nevertheless, despite this natural variability, the silencing we observed was strong when compared to the control for each time point. and consistent in all experiments and repetitions.

Bottom Line:
Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW.The body of treated insects showed inhibition of sclerotization, leading eventually to death.Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing.

ABSTRACTThe African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.